Enveloped viruses release their nucleocapsids into the cytoplasm by merging their membrane with the cell membrane. The majority of these viruses is internalized by cells and fuses with endosomes. The elucidation of the molecular mechanisms of virus-endosome fusion and its regulation has been hampered by the highly dynamic nature of endosomes and the lack of access to these compartments. We have developed non- invasive imaging techniques that permit the time-resolved visualization of the critical steps of virus entry, from hemifusion (lipid transfer), to small pore formation (release o small content markers) and pore enlargement (release of the capsid). The recently implemented imaging assay enables (i) measurements of the pH in virus- carrying endosomes and (ii) detection of the resulting fusion events. We propose to apply these imaging and other approaches to define the mechanism of entry of the Avian Sarcoma and Leukosis Virus (ASLV), which is an excellent model for elucidating the entry pathways used by disparate viruses. The two-step triggering of ASLV-endosome fusion - binding to a cognate receptor and exposure to low pH - permit an unprecedented control over the virus entry process. Our pilot data suggest that, strikingly, the ASLV fusion with early acidic endosomes is restricted in some cell types and occurs in late endosomal compartments. These findings imply that ASLV fusion is regulated by cellular factors and that this virus may undergo hemifusion or form a small fusion pore in early endosomes, while relying on host factors to drive the energetically unfavorable step of pore enlargement. To test this hypothesis, we will: (1) investigate the spatio-temporal regulation of ASLV fusion; (2) examine the determinants of ASLV pore enlargement; and (3) define the role of endosomal lipids in ASLV fusion. These studies will provide critical insights into the ASLV fusion and its regulation by host factors. Knowledge of the mechanisms of ASLV fusion will offer a conceptual framework for studies of human viruses, such as HIV-1, Hepatitis C virus and Ebola virus, which enter host cells via the endocytic pathway and rely on a multitude of host factors.